This invention relates to bio-chemical analysis. More particularly, it relates to miniaturized unitary analysis apparatus in which a reaction chamber, a heater and an analysis chamber are formed to permit rapid thermo-cycling and analysis of bio-chemical reactions.
Genetic testing of DNA and related materials is an integral part of clinical, commercial and experimental biology. In the medical field, for example, genetic tests are critical for effective treatment of cancer and inherited diseases. Oncologists use genetic tests to obtain the cytogenetic signature of a malignancy which in turn guides the choice of therapy and improves the accuracy of prognoses. Similarly, monitoring frequency and type of mutations persisting after chemotherapy or radiation therapy provides quick and accurate assessment of the impact of the therapy. Perhaps the most important application of molecular diagnosis in oncology is the emerging possibility of using antisense genetic therapy to combat tumor growth.
Inherited diseases occur when a person inherits two copies of a defective version of a gene. (A version of a gene is referred to hereinafter as an allele.) Genetic tests can determine which genes and alleles are responsible for a given disease. Once the gene is identified, further testing can identify carriers of the allele and aid researchers in designing treatments for the disease.
Most genetic tests begin by amplifying a portion of the DNA molecule found within a sample of biological material. Amplification is made practical by the polymerase chain reaction (PCR) wherein a DNA synthesizing enzyme (polymerase) is used to make multiple copies of a targeted segment of DNA. By repeating the polymerase copying process, many copies of the targeted segment are produced. For example, thirty (30) repetitions can produce one million (1,000,000) molecules from a single molecule.
Changing the temperature (thermo-cycling) of the test sample drives the PCR reaction. The optimum thermo-cycle varies, however, depending on the material amplified and/or the result sought. In a typical PCR process the sample is heated and cooled to three different target temperatures and is maintained at each temperature for a length of time sufficient for the sample to undergo the desired change. The thermo-cycle begins with heating the sample to about 95xc2x0 C. to separate the double strands and make them accessible as templates for polymerase replication. Cooling to about 55xc2x0 C. allows the polymerase initiators (primers) to hybridize with their target DNA segments. Control of the temperature during the hybridization process is critical for accurate hybridization of the primer to the DNA. Heating from 55xc2x0 C. to about 72xc2x0 C. is necessary for efficient performance of the polymerase enzyme. At the appropriate temperature, the polymerase reaction catalyzes the elongation of new DNA complementary in nucleotide sequence to the target DNA. At the end of the elongation reaction, heating the solution to about 95xc2x0 C. causes the newly formed double-stranded DNA to separate into single strands, thus providing templates for another round of PCR amplification.
Current thermo-cycling methods are complex, time consuming and costly. One thermo-cycling device (known as the MJ Research DNA engine) comprises a surface on which are formed micro-wells and under which rests a thermo-electric block for heating and cooling biological material placed in the wells. However, this device takes about one and one-half (1.5) minutes to perform each cycle, even when using a simplified two temperature format. The device thus requires approximately forty-five minutes to perform a thirty cycle run.
Various devices using capillary tubes can perform a thirty cycle run in from ten to thirty minutes. These devices require loading and unloading samples to and from the tubes, sealing the tubes and then exposing the tubes to forced air heating. When the loading and unloading steps are included, these procedures may consume as much as two hours of laboratory time. These procedures also require relatively skilled technicians who can make and load 2-D gels and accurately handle microliter volumes of reagents.
One conventional thermo-cycler uses forced water circulation to heat and cool vessels immersed in a water bath. Three or more reservoirs hold water at different temperatures and rapid pumps and valves bring water from the reservoirs into the bath to produce a huge thermal mass which heats or cools the material in the vessels. Another device used in PCR processes (see European Patent No. 381501) utilizes a flexible bag-like structure with an inner system of chambers. The DNA sample and reagent fluids are loaded into the chambers and the bag is placed on a hot plate for thermo-cycling. After thermo-cycling, the bag is squeezed with external rollers to move the fluid into chambers containing detection reagents.
In all the prior art methods, a significant amount of thermo-cycle time is consumed by ramp periods wherein the temperature of the biological material is changed from one target temperature to the next. The length of each ramp period is a function of both the thermo-cycling equipment and the volume of material heated. The prior art methods generally require first heating the test chamber which then transfers heat to the material contained therein. The thermal mass of the test chamber and volume of material in the test chamber produces high thermal inertia and poor heat-loss surface area to volume ratios. Furthermore, the reagents used in the procedure are expensive and their volume (even if only in the fifty microliter range) can make the procedure prohibitively expensive.
Prior methods pose further time limitations by typically generating only one thermo-cycle at a time. Thus, processing multiple samples at the optimum thermo-cycle of each requires processing the samples one after the other (serial processing). Serial processing may be avoided by using multiple thermo-cyclers, but this approach consumes capital, energy and laboratory space.
Multiple DNA samples can be processed simultaneously using parallel processing. The most common parallel processing technique involves grouping several DNA samples together and subjecting them to a common thermo-cycle. However, the common cycle is necessarily a compromise among the optimum cycles and time savings are thus achieved at the expense of quality of results.
After the material is amplified using one of the foregoing methods it is subjected to further testing. An example of a test which may require PCR amplification as a first step is one which detects the presence of an allele found within a DNA sample. An allele specific oligonucleotide (a substance which will react with the allele and referred to as an ASO hereinafter) is deposited at a known location on a test strip. The DNA sample is labeled using conventional methods, such as by mixing the sample with a fluorescent or radioactive material. The test strip is exposed to the DNA sample. If the sought allele is present it binds with the ASO on the test strip along with the labeling material. The test strip may then be examined to determine if the point where the ASO is deposited exhibits characteristics of the label.
In accordance with the present invention, apparatus is provided which incorporates reaction chambers, heaters and analysis chambers into a miniature self-contained compact structure or cassette. The reaction chambers and heaters are used to simultaneously process extremely small, multiple samples of biological matter at their optimum thermo-cycles. The analysis chambers are used to analyze the results of the reactions. The biological material is maintained within the same cassette for both the PCR and the subsequent analysis, thus many intermediate procedural steps and much of the overall processing time is eliminated or reduced. By using extremely small samples of biological matter and incorporating heaters directly into the cassette structure, thermo-cycling is performed with extremely short ramp periods. The small samples also reduce the overall cost of performing the procedure by minimizing the quantity of expensive reagents required. The cassette is pre-loaded with reagents and analytical materials to further reduce the complexity of the steps which must be performed in the laboratory or clinic, thus making the apparatus amenable for use with automated processes.